Title The natural adhesive of the goose barnacle Lepas anatifera

190 Pages·2017·8.07 MB·English

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The natural adhesive of the goose barnacle Lepas anatifera:
Title The functional morphology and chemistry of the adhesive
gland and an investigation of the adhesive proteins
Author(s) Jonker, Jaimie-Leigh
Publication
2013-12-25
Date
Item record http://hdl.handle.net/10379/5041
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The natural adhesive of the goose barnacle
Lepas anatifera:
The functional morphology and chemistry of the adhesive
gland and an investigation of the adhesive proteins

by

Jaimie-Leigh Jonker

Supervisor: Dr Anne Marie Power
School of Natural Sciences
Zoology
National University of Ireland, Galway
September 2013

Table of Contents
Declaration ................................................................................................................... v
Acknowledgements ..................................................................................................... vii
Abstract ....................................................................................................................... ix
1 General introduction .................................................................................................. 1
1.1 Underwater adhesion ........................................................................................... 1
1.1.1 Bioadhesion .................................................................................................. 1
1.1.2 Bioadhesion underwater ............................................................................... 2
1.2 Barnacle adhesion ............................................................................................... 4
1.2.1 Barnacles ...................................................................................................... 4
1.2.2 The barnacle adhesive apparatus ................................................................... 5
1.2.3 Barnacle Adhesive ........................................................................................ 7
1.2.4 Mechanism of adhesion ................................................................................ 7
1.3 Adhesive biomaterials ......................................................................................... 9
1.3.1 Bioadhesives as biomaterials ...................................................................... 10
1.4 Aims ................................................................................................................. 12
1.5 References......................................................................................................... 14
2 The collection and long term cultivation of the adult goose barnacle Lepas anatifera19
2.1 Introduction ....................................................................................................... 19
2.1.1 Distribution and life cycle of Lepas anatifera ............................................. 19
2.1.2 Cultivating stalked barnacles ...................................................................... 20
2.1.3 Aims........................................................................................................... 22
2.2 Methods and results: sampling........................................................................... 23
2.2.1 Onshore sampling ....................................................................................... 23
2.2.2 Offshore sampling ...................................................................................... 25
2.3 Methods and results: cultivation ........................................................................ 27
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2.3.1 Culture conditions....................................................................................... 27
2.3.2 Cultivation of larvae ................................................................................... 29
2.4 Discussion ......................................................................................................... 31
2.5 References ......................................................................................................... 33
3 Unusual adhesive production system in the barnacle Lepas anatifera ....................... 35
3.1 Introduction ....................................................................................................... 35
3.2 Materials and methods ....................................................................................... 38
3.3 Results .............................................................................................................. 41
3.3.1 Morphology of the glandular apparatus ....................................................... 41
3.3.2 Chemistry of the gland cell ......................................................................... 44
3.3.3 Assays for post-translational modifications ................................................. 47
3.3.4 Ultrastructure of the canal network ............................................................. 49
3.3.5 Features of the principal canal and secretion ............................................... 51
3.4 Discussion ......................................................................................................... 54
3.5 References: ........................................................................................................ 60
4 Scanning electron microscopy and spectroscopic analyses of the adhesive of Lepas
anatifera ..................................................................................................................... 65
4.1 Introduction ....................................................................................................... 65
4.2 Materials and methods ....................................................................................... 68
4.2.1 Sample collection ....................................................................................... 68
4.2.2 SEM-EDS ................................................................................................... 69
4.2.3 TEM-EDS .................................................................................................. 70
4.2.4 Raman spectroscopy ................................................................................... 70
4.2.5 FTIR spectroscopy ...................................................................................... 70
4.3 Results .............................................................................................................. 71
4.3.1 SEM of peduncle tissues ............................................................................. 71
4.3.2 SEM-EDS of adhesive gland....................................................................... 72
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4.3.3 SEM-EDS of barnacle adhesive .................................................................. 73
4.3.4 TEM-EDS of peduncle tissues .................................................................... 75
4.3.5 Raman spectra of barnacle adhesive ............................................................ 75
4.3.6 FTIR spectroscopy...................................................................................... 81
4.4 Discussion ......................................................................................................... 83
4.5 References......................................................................................................... 89
5 The adhesive proteins of Lepas anatifera ................................................................. 93
5.1 Introduction ....................................................................................................... 93
5.2 Methods ............................................................................................................ 95
5.2.1 Collection of adhesive ................................................................................ 95
5.2.2 Solubilisation of adhesive ........................................................................... 95
5.2.3 One-dimensional SDS-PAGE ..................................................................... 96
5.2.4 Mass spectrometry ...................................................................................... 97
5.2.5 Degenerate primer design ........................................................................... 98
5.2.6 RNA extraction, cDNA synthesis and PCR ................................................. 99
5.2.7 RACE PCR .............................................................................................. 101
5.2.8 Ligation, transformation and cloning ........................................................ 101
5.2.9 Sequence analysis ..................................................................................... 102
5.3 Results ............................................................................................................ 103
5.3.1 Solubilisation and separation .................................................................... 103
5.3.2 Analysis of protein bands with mass spectrometry .................................... 104
5.3.3 Similarities between L. anatifera and D. fascicularis adhesive proteins .... 106
5.3.4 Amplification of de novo fragments, including RACE PCR ...................... 107
5.4 Discussion ....................................................................................................... 112
5.5 References....................................................................................................... 117
6 Barnacle adhesive proteins: conservation and localisation ...................................... 121
6.1 Introduction ..................................................................................................... 121
iii
6.1.1 Conservation of barnacle adhesive proteins ............................................... 121
6.1.2 Conservation of adhesive proteins from other marine animal models ........ 121
6.1.3 Barnacle adhesive protein production ....................................................... 123
6.1.4 Aims ......................................................................................................... 124
6.2 Materials and methods ..................................................................................... 125
6.2.1 Immunohistochemistry ............................................................................. 125
6.2.2 Degenerate primer design ......................................................................... 125
6.2.3 RNA extraction, cDNA synthesis and PCR ............................................... 127
6.2.4 Ligation, transformation and cloning ........................................................ 129
6.2.5 Sequence analysis ..................................................................................... 129
6.3 Results ............................................................................................................ 130
6.3.1 Immunohistochemistry ............................................................................. 130
6.3.2 Sequence alignment and primer design ..................................................... 134
6.3.3 Amplification of homologues.................................................................... 142
6.4 Discussion ....................................................................................................... 147
6.5 References ....................................................................................................... 151
7 General discussion ................................................................................................. 155
7.1 Research summary .......................................................................................... 155
7.2 Adhesive production system ............................................................................ 155
7.3 Mechanisms of adhesion.................................................................................. 156
7.4 Conservation of adhesive components ............................................................. 159
7.5 Conclusions ..................................................................................................... 160
7.6 References ....................................................................................................... 162
8 References ............................................................................................................. 165

iv
Declaration

I hereby declare that this thesis is my own original research work, conducted under the
supervision of Dr Anne Marie Power. This work has not previously been submitted for
the award of any other degree or diploma.

The work described in chapter 2, the collection and cultivation of goose barnacles, was
conducted alongside another student and described in: McEvilly, P. (2011). A look at
some components of the adhesive of the pedunculate barnacles Dosima fascicularis,
Lepas anatifera, Conchoderma auritum, Lepas pectinata and Pollicipes pollicipes.
(Research Masters), National University of Ireland, Galway, Galway. This work was
divided equally between myself and Paul McEvilly.

Chapter 3 has previously been published as: Jonker, J.-L., von Byern, J., Flammang, P.,
Klepal, W., & Power, A. M. (2012). Unusual adhesive production system in the
barnacle Lepas anatifera: An ultrastructural and histochemical investigation. Journal of
Morphology, 273(12), 1377-1391. This work was performed and written by myself
with co-authors acting as teachers and supervisors of histology and histochemistry (J.
von Byern), immunohistochemistry (P. Flammang) and ultrastructure (W. Klepal).

Chapter 4 was conducted with the help and supervision of Dr Liam Morrison, for his
expertise in scanning electron microscopy, energy dispersive spectrometry and Raman
spectroscopy.

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vi
Acknowledgements

First and foremost, I want to thank my supervisor Anne Marie Power for providing me
with the opportunity to travel to Ireland and complete a PhD at NUI Galway. She and
the rest of the Zoology department of NUI Galway, especially the technical staff,
provided invaluable support throughout my time there.

Second, I would like to thank my friend and colleague Paul McEvilly for being my
constant companion throughout the first half of my PhD and continuing to provide
support and optimism from afar. Further thanks go out to all of the friends I made
during the course of my PhD, many of whom were also my teachers for various aspects
of this multidisciplinary work; you are too numerous to name but I truly could not have
made it to the end without you all so thank you.

Many colleagues provided me with vital skills and advice over my years at NUI
Galway, choosing to give up their time and energy to teach me and help find solutions
when things went wrong. Thanks to Professor Waltraud Klepal, Dr Janek von Byern
and Vanessa Zheden for teaching and looking after me so well in Vienna, to Ingo
Grunwald and his teaching assistants for my time in Bremen and to Professor Patrick
Flammang and Dr Elise Hennebert for my short time in Mons.

Many thanks to Pearce Lalor and Mark Canning of the NUI Galway Anatomy
department, for taking such an interest in my work even though they didn’t have to, and
the rest of the Anatomy department for allowing me to work there and helping me when
I needed it. Dr Liam Morrison chose to take an interest in my studies and was full of
enthusiasm and ideas, giving me his time and skills and providing me with further
contacts, all of which gave my PhD a necessary boost. Dr Florence Abram of the NUI
Galway Microbiology department not only chose to teach me, but gave me support and
friendship and I am extremely grateful for her help.

Finally, to the all the people out there, scattered across various countries, that gave me
advice, information, support, friendship, or just a laugh, I would like to thank you for
being a part of this huge task that has been my life for the last four years.
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